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Notes: The authors of this paper studied the targeting mode of the Parkinson disease-associated kinase Pink1. They constructed a number of truncation and deletion mutants in the pGEM®-4Z Vector, and verified the identity of these clones using next-generation sequencing. The authors then expressed radiolabeled versions of the various Pink1 constructs using the TNT® Coupled Rabbit Reticulocyte Lysate System. These labeled proteins were used in mitochondrial localization studies. (4563)

Notes: Oxidative damage has been associated with a range of age-related neurological conditions. In this study, the effect of mRNA oxidation was investigated. A direct correlation was observed between the extent of oxidation and the frequency of translation errors. The authors excised the firefly luciferase (luc2) gene from the pGL4.14 Vector, attached a FLAG tag to the 5´ terminus and a Myc tag to the 3´ terminus, and subcloned the gene into a pGEM-4Z Vector that had been modified to append a poly(A) sequence. The construct was transfected into HEK293 cells, which were then cultured in the presence of an oxidizing agent. The occurrence of truncated protein fragments and short peptides increased in the presence of the oxidizing agent in a concentration-dependent manner. The effects of oxidation of mRNA were also investigated in in vitro translation experiments using mRNA treated with an iron-ascorbate mixture and hydrogen peroxide. Translation in vitro was performed using rabbit reticulocyte lysate supplemented with protease inhibitors. The translation products were detected using anti-FLAG and anti-c-Myc antibodies. (3630)

Notes: The authors used Xenopus laevis oocytes to show that unliganded thyroid hormone receptor (TR) recruits N-CoR (nuclear receptor corepressor) to modulate metamorphosis. To study this influence, the cytoplasm of stage VI oocytes from X. laevis was injected with the indicated mRNAs [TR, retinoic acid receptor (RXR) and FLAG-tagged N-CoR]. The reporter plasmid TRE-Luc (0.33 ng/oocyte; thyroid hormone response elements from a Xenopus promoter driving expression of the firefly luciferase gene) and the control vector phRG-TK (0.03 ng/oocyte) were co-injected into the germinal vesicle (nucleus) after mRNA injection. After overnight incubation at 18°C, oocyte lysates were prepared by lysing six oocytes in 90µL 1X Passive Lysis Buffer. The Dual-Luciferase® Reporter Assay System was then used to assay 7µl of the lysate. The researchers also used an expression vector (based on the pGEM®-4Z Vector) containing the 5’ and 3’ untranslated regions of the X. laevis beta-globin gene flanking the multiple cloning site. (3088)

Notes: The Wizard® Lambda DNA Purification System was used to isolate phage for the Sphingomonas paucimobilis and Flavobacterium sp. isolates. The isolated DNA was used for determination of the phage genome size by restriction digestion. The Wizard® Genomic DNA Purification Kit was used to isolate the genomic DNA from these bacterial species as well. The authors note that the DNA isolated with the Wizard® Genomic Kit was as pure as that purified by a cesium chloride gradient. Loading of the DNA isolated with the Genomic Kit onto cesium chloride gradients produced only a single sharp band after centrifugation. The isolated DNA was used for dot blot hybridizations. (0979)

Notes: Sixty patients were screened for autoantibodies to IA-2 (a protein tyrosine phosphatase-like molecule), IA-2ic (the intracellular fragment of IA-2) and GAD65 (glutamic acid decarboxylase) using RIAs. Full-length human cDNAs were cloned into the pGEM®-4Z Vector and expressed in the TNT® Coupled Reticulocyte Lysate System in the presence of [35S]methionine. Incorporated radiolabel was determined by precipitation and scintillation counting. Immunoprecipitation was performed with 5µl of serum and counted on a multiwell counter. Antibodies to IA-2 or GAD65 were detected in 87% of newly diagnosed type 1 diabetic patients and in all 56 prediabetic samples from 11 twins. The frequencies of antibodies to IA-2ic were similar in type 1 diabetics. (1763)

EMBO J.15, 4613-4628.
The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.1996

MacNeill, S.A., Moreno, S., Reynolds, N., Nurse, P. and Fantes, P.A.

Notes: HA and Myc epitope tags were introduced at the C-terminus of Cdc1 and Cdc27 by oligo-directed mutagenesis. Cdc1tag and Cdc27tag were cloned into the pGEM®-4Z Vector and expressed in the TNT® T7 Coupled Reticulocyte Lysate System ± [35S]methionine. In GST coimmunoprecipitation assays, Cdc1 interacts with GST-Pol3, and the GST-Pol3 binds preferentially to a truncated Cdc1 translation product. To confirm an interaction observed between Cdc1 and Cdc27, the TNT®-expressed proteins were mixed together and co-immunoprecipitated using the anti-Cdc27 antibodies Using EGS (ethylene glycol-bis-succinimidylsuccinate) labeled, in vitro translated Cdc1 and Cdc27 were cross-linked. (1771)

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